Almost half a million new cases of colorectal cancer (CRC) worldwide are diagnosed each year. Studies from our lab and others have shown that CRC initiating cells (CCIC) are important for CRC formation. In contrast to commonly used CRC cell lines, CCIC serially maintain tumors with the pathological and molecular markers of the primary CRCs from which they were derived. However, the mechanism of CCIC tumor formation, and how it differs from that used by commonly used CRC cell lines to form tumors, is poorly characterized. We recently derived new CCIC lines and made several novel findings. These include data that (1) CCIC have 30X+ higher NOTCH signaling levels than commonly used CRC cell lines, (2) NOTCH signaling is critical for CCIC self-renewal and tumor formation and (3) CCIC use a NOTCH driven mechanism to mitose asymmetrically (similar to the mechanism used by normal colon stem cells to form colon crypts) and generate distinct daughter cells. This is the first example of CRC cell asymmetric mitosis. Asymmetric mitosis is critical for leukemia CIC tumor formation and self-renewal and is likely to play a similar role for CCIC. Since commonly used CRC cell lines do not mitose asymmetrically this study will provide unique insights into the mechanism of CCIC tumor formation and how it differs from that used by commonly used CRC cell lines. The overall goal of this proposal is to understand the mechanism that regulates the balance between colon cancer initiating cell symmetric and asymmetric mitosis and the role of each in CCIC self-renewal and tumor formation. Based on the role of NOTCH in normal colon stem cell asymmetric mitosis and our preliminary data we hypothesize that NOTCH, NUMB and HGF/MET critically regulate colon cancer initiating cell asymmetric/symmetric mitosis, tumor formation and self-renewal. We propose AIM 1 Identify the mechanism of NOTCH driven CCIC asymmetric mitosis and tumor formation and AIM 2 Test hypotheses that NUMB and HGF/MET regulate CCIC symmetric and asymmetric mitosis PUBLIC HEALTH RELEVANCE: CRC is the 2nd leading cause of cancer death in the United States. When CRC has spread to other organs, 5- year survival is 10-15%. Because colon cancer initiating cells (CCIC) are linked to tumorigenesis and metastasis but are poorly characterized, understanding the mechanisms of CCIC self-renewal, asymmetric mitosis and metastasis are important innovative approaches to discover better CRC chemoprevention and chemotherapy targets.